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Abstract:

The invention relates to plant extracts and bioactive molecules derived
from the plant genus Acronychia and their use as antioxidants,
antibacterials, anthelmintic, anti-inflammatories, cancer
chemopreventatives, food additives and fragrance components in
pharmaceuticals, nutraceuticals, foods and cosmetics.

Claims:

1. A method of treating or preventing a bacterial infection, helmintic
infection, a cell proliferative disorder, an inflammatory disease or
disorder or a disorder related to oxidative stress comprising
administering to a subject an effective amount of an extract from the
fruit of an Acronychia species.

2. A method of treating or preventing a bacterial infection comprising
administering to a subject an effective amount of a compound of formula
(I): ##STR00014## wherein A is an aryl group or a heteroaryl group; Z
is selected from --O--, --S-- and --NR4--; Y is selected from a
covalent bond and --(CH2)p--; R1 is selected from
--C5-C20alkyl, --C5-C20alkenyl,
--C5-C20alkynyl and --C3-C8cycloalkyl; R2 is
selected from hydrogen, --C1-C6alkyl, --C2-C6alkenyl,
--C2-C6alkynyl, --C3-C8cycloalkyl, hydroxy,
--OC1-C6alkyl, --OC2-C6alkenyl,
--OC2-C6alkynyl, --OC3-C8cycloalkyl, thiol,
--SC1-C6alkyl, --SC2-C6alkenyl,
--SC2-C6alkynyl, --SC3-C8cycloalkyl,
--NR4C1-C6alkyl, --NR4C2-C6alkenyl,
--NR4C2-C6alkynyl and --NR4C3-C8cycloalkyl;
R3 is selected from --CO2H or an isosteric equivalent of a
carboxy group; R4 is selected from hydrogen and
--C1-C6alkyl; and p is an integer from 1 to 10; or a
pharmaceutically salt thereof.

3. A method according to claim 2 wherein the compound is a compound of
formula (1A): ##STR00015## Y is selected from a covalent bond and
--(CH2)p--; R1 is selected from --C5-C20alkyl,
--C5-C20alkenyl and --C3-C8cycloalkyl; R2 is
selected from hydrogen, --C1-C6alkyl, --C2-C6alkenyl,
--C2-C6alkynyl, --C3-C8cycloalkyl, hydroxy,
--OC1-C6alkyl, --OC2-C6alkenyl,
--OC2-C6alkynyl and --OC3-C8cycloalkyl; R3 is
selected from --CO2H or an isosteric equivalent of a carboxy group;
and p is an integer from 1 to 10; or a pharmaceutically salt thereof.

4. A method according to claim 2 wherein R1 is C5-C15
alkenyl having 1 to 3 double bonds.

5. A method according to claim 4 wherein R1 is farnesyl.

6. A method according to claim 2 wherein R2 is hydrogen or
--OC1-C3 alkyl.

7. A method according to claim 2 wherein R3 is --CO2H.

8. A method according to claim 2 wherein Y is --(CH2)p--.

9. A method according to claim 8 wherein p is 1 to 4.

10. A method according to claim 1 wherein the bacterial infection is
caused by a Gram positive or Gram negative bacteria.

13. A method according to claim 1 wherein the Acronychia species is
selected from A. acidula, A. aberrans, A. acronychioides and A.
crassipetala.

14. A method according to claim 13 wherein the Acronychia species is A.
acidula.

15. A compound of formula (II): ##STR00016## wherein A is an aryl group
or a heteroaryl group; Z is selected from --O--, --S-- and --NR4--;
Y is --(CH2)p--; R1 is selected from
--C5-C20alkyl, --C5-C20alkenyl,
--C5-C20alkynyl and --C3-C8cycloalkyl; R2 is
selected from --C1-C6alkyl, --C2-C6alkenyl,
--C2-C6alkynyl, --C3-C8cycloalkyl, hydroxy,
--OC1-C6alkyl, --OC2-C6alkenyl,
--OC2-C6alkynyl, --OC3-C8cycloalkyl, thiol,
--SC1-C6alkyl, --SC2-C6alkenyl,
--SC2-C6alkynyl, --SC3-C8cycloalkyl,
--NR4C1-C6alkyl, --NR4C2-C6alkenyl,
--NR4C2-C6alkynyl and --NR4C3-C8cycloalkyl;
R3 is selected from --CO2H or an isosteric equivalent of a
carboxy group; R4 is selected from hydrogen and
--C1-C6alkyl; and p is an integer from 1 to 10; or a
pharmaceutically salt thereof.

16. A compound according to claim 15 wherein the compound is a compound
of formula (IIA): ##STR00017## Y is --(CH2)p--; R1 is
selected from --C5-C20alkyl, --C5-C20alkenyl and
--C3-C8cycloalkyl; R2 is selected from
--C1-C6alkyl, --C2-C6alkenyl,
--C2-C6alkynyl, --C3-C8cycloalkyl, hydroxy,
--OC1-C6alkyl, --OC2-C6alkenyl,
--OC2-C6alkynyl and --OC3-C8cycloalkyl; R3 is
selected from --CO2H or an isosteric equivalent of a carboxy group;
p is an integer from 1 to 10; or a pharmaceutically salt thereof.

17. A compound according to claim 15 wherein R1 is C5-C15
alkenyl having 1 to 3 double bonds.

22. A compound according to claim 15 wherein Y is --(CH2)p--
and p is 1 to 6.

23. A compound according to claim 22 wherein p is 1 to 4.

24. A pharmaceutical composition comprising an extract from the fruit of
an Acronychia species or a compound of formula (I) as defined in claim 2,
or formula (II) as defined in claim 15, and a pharmaceutically acceptable
salt thereof.

25. A fragrance composition comprising an extract from the fruit of an
Acronychia species, said extract comprising at least one of:
1-ethenyl-1-methyl-2,4-bis(1-methylethenyl)cyclohexane,
2,6-di-tert-butylbenzoquinone, 2,5-di-tert-butyl-1,4-benzoquinone,
Tetracontane-1,40-diol, and
2,2,5,5-tetramethyl-bicyclo[6.3.0]undec-1(8)-enone.

26. A cosmetic or fragrance composition comprising an extract from the
fruit of an Acronychia species, wherein the extract is obtained by a
method comprising initial water or alcohol extraction and a subsequent
ethyl acetate extraction.

27-32. (canceled)

33. A method according to claim 2 wherein the bacterial infection is
caused by a Gram positive or Gram negative bacteria.

Description:

FIELD OF THE INVENTION

[0001] The invention relates to plant extracts and bioactive molecules
derived from the plant genus Acronychia and their use as antioxidants,
antibacterials, anthelmintic, anti-inflammatories, cancer
chemopreventatives, food additives and fragrance components in
pharmaceuticals, nutraceuticals, foods and cosmetics.

BACKGROUND OF THE INVENTION

[0002] Biodiscovery is a field of endeavour that investigates and screens
for bioactive natural products from natural environments such as plants,
microorganisms, soils and marine life. In biodiscovery, biological
materials are screened for molecules having properties which may be of
therapeutic benefit for use in treatment of humans or animals, for use in
cosmetic compositions, for use as food additives or fragrance components.

SUMMARY OF THE INVENTION

[0003] The present invention is predicated in part on the discovery that
extracts of the fruit of Acronychia species have potent antibacterial,
anthelmintic, antioxidant, anti-inflammatory and/or anti-cancer activity.

[0004] In a first aspect of the invention there is provided a method of
treating or preventing a bacterial infection comprising administering to
a subject an effective amount of an extract from an Acronychia species.

[0005] In a further aspect of the invention there is provided a method of
treating or preventing a helminthic infection in a subject comprising
administering an effective amount of an extract from an Acronychia
species.

[0006] In another aspect of the invention there is provided a method of
treating or preventing a cell proliferative disorder comprising
administering to a subject an effective amount of an extract from an
Acronychia species.

[0007] In another aspect of the invention there is provided a method of
treating or preventing an inflammatory disease or disorder comprising
administering to a subject an effective amount of an extract from an
Acronychia species.

[0008] In a further aspect of the invention there is provided a method of
treating or preventing a disease or disorder related to oxidative stress
comprising administering to a subject an effective amount of an extract
from an Acronychia species.

[0009] In yet another aspect of the invention there is provided a method
of treating or preventing a bacterial infection comprising administering
to a subject an effective amount of a compound of formula (I):

##STR00001##

[0010] wherein A is an aryl group or a heteroaryl group;

[0011] Z is selected from --O--, --S-- and --NR4--;

[0012] Y is selected from a covalent bond and --(CH2)p--;

[0013] R1 is selected from --C5-C20alkyl,
--C5-C20alkenyl, --C5-C20alkynyl and
--C3-C8cycloalkyl;

[0024] R3 is selected from --CO2H or an isosteric equivalent of
a carboxy group;

[0025] R4 is selected from hydrogen and --C1-C6alkyl;

[0026] and p is an integer from 1 to 10; or a pharmaceutically salt
thereof.

[0027] In a further aspect of the invention there is provided a
pharmaceutical composition comprising an extract from Acronychia species
or a compound of formula (I) or formula (II) and a pharmaceutically
acceptable salt thereof.

[0028] In yet another aspect of the invention there is provided a flavour
or fragrance composition comprising an extract from an Acronychia
species, said extract comprising at least one of: [0029]
1-ethenyl-1-methyl-2,4-bis(1-methylethenyl)cyclohexane, [0030]
2,6-di-tert-butylbenzoquinone, [0031] 2,5-di-tert-butyl-1,4-benzoquinone,
[0032] Tetracontane-1,40-diol, and [0033]
2,2,5,5-tetramethyl-bicyclo[6.3.0]undec-1(8)-enone.

[0034] In yet another aspect of the invention, there is provided a
cosmetic, food or fragrance composition comprising an extract from an
Acronychia species, wherein the extract is obtained by a method
comprising initial water or alcohol extraction and a subsequent ethyl
acetate extraction.

[0035] In a further aspect of the invention there is provided a use of an
extract from an Acronychia species as a fragrance or flavour component in
a food or other composition.

[0036] In another aspect of the invention there is provided the use of an
extract of an Acronychia species as a food additive, a fragrance
component or an antioxidant, an anti-inflammatory or an antibacterial
component of a cosmetic composition.

DETAILED DESCRIPTION OF THE INVENTION

[0037] The plant genus Acronychia is a member of the citrus family
(Ruticeae) and comprises approximately 50 species that occur naturally in
Australia, the Pacific Islands, Malesia and Asia. Extracts from any of
these species may be used in the invention. In some embodiments the
extract is obtained from Acronychia species native to Australia including
A. aberrans, A. acidula, A. acronychioides, A. acuminate, A. baeuerlenii,
A. chooreechillum, A. crassipetala, A. eungellensis, A. imperforate, A.
laevis, A. littoralis, A. oblongifolia, A. octanara, A. parviflora, A.
pauciflora, A. pubescens, A. species (Batavia Downs), A. suberosa, A.
vestita and A. wilcoxiana. In particular embodiments, the extract is
obtained from A. acidula, A. aberrans, A. acronychioides or A.
crassipetala, especially A. acidula.

[0038] The extract may be obtained from any part of the plant such as the
fruit, the seed, the bark, the leaf, the flower, the roots and the wood.
In particular embodiments, the extract is obtained from the fruit of the
plant.

[0039] For example, biomass obtained from the fruit of the plant is
subject to initial solvent extraction, for example with a polar solvent,
for example water or an alcohol such as methanol or ethanol. The initial
extraction is then concentrated and diluted with water and subject to
extraction with a second solvent, for example, ethyl acetate. The solvent
samples from the second extraction are pooled and subject to separation
by preparative HPLC fractionation. The fractions are analysed by
analytical HPLC and pooled according to the retention time of compounds
found in the samples. The pooled fractions are weighed, bioassayed and
analysed by analytical HPLC. Further fractionation using one or more
preparative HPLC is performed to isolate specific compounds. Each
compound is bioassayed and its structure identified by UV, NMR and mass
spectrometric (LC and GC) techniques.

[0040] In one aspect of the invention there is provided a method of
treating or preventing a bacterial infection comprising administering to
a subject an effective amount of an extract from an Acronychia species.

[0043] In some embodiments, specific compounds are obtained from the
extract and used in the method of treating or preventing bacterial
infections, or derivatives or analogues of such compounds are used. For
example, suitable compounds obtained from the extract, derivatives or
analogues of such compounds are compounds of formula (I):

##STR00003##

[0044] wherein A is an aryl group or a heteroaryl group;

[0045] Z is selected from --O--, --S-- and --NR4--;

[0046] Y is selected from a covalent bond and --(CHDp--;

[0047] R1 is selected from --C5-C20alkyl,
--C5-C20alkenyl, --C5-C20alkynyl and
--C3-C8cycloalkyl;

[0070] The term "alkenyl" refers to optionally substituted unsaturated
linear or branched hydrocarbon groups, having 2 to 20 carbon atoms and
having at least one double bond. Where appropriate, the alkenyl group may
have a specified number of carbon atoms, for example, C2-C6
alkenyl which includes alkenyl groups having 2, 3, 4, 5 or 6 carbon atoms
in linear or branched arrangements. Non-limiting examples of alkenyl
groups include, ethenyl, propenyl, isopropenyl, butenyl, s- and
t-butenyl, 3-methylbut-2-enyl, pentenyl, hexenyl, hept-1,3-dienyl,
hex-1,3-dienyl, hexa-1,3,5-trienyl, heptenyl, octenyl,
3,7-dimethyl-octa-2,6-dienyl, nonenyl, decenyl, undecenyl and farnesyl
and the like.

[0071] The term "alkynyl" refers to optionally substituted unsaturated
linear or branched hydrocarbon groups, having 2 to 20 carbon atoms and
having at least one triple bond. Where appropriate, the alkynyl group may
have a specified number of carbon atoms, for example, C2-C6
alkynyl groups have 2, 3, 4, 5 or 6 carbon atoms in linear or branched
arrangements. Non-limiting examples of alkynyl groups include ethynyl,
propynyl, butynyl, pentynyl, hexynyl and the like.

[0072] The terms "cycloalkyl" refer to optionally substituted saturated or
unsaturated monocyclic, bicyclic or tricyclic carbon groups. Where
appropriate, the cycloalkyl group may have a specified number of carbon
atoms, for example, C3-C6 cycloalkyl is a carbocyclic group
having 3, 4, 5 or, 6 carbon atoms. Non-limiting examples may include
cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl,
cyclohexenyl, cyclohexadienyl, cycloheptyl and cyclooctyl and the like.

[0073] "Aryl" means a C6-C10 membered monocyclic or bicyclic
carbocyclic ring system having up to 7 atoms in each ring, wherein at
least one ring is aromatic. Examples of aryl groups include, but are not
limited to, phenyl, naphthyl and tetrahydronaphthyl. The aryl may
comprise 1-2 benzene rings. If two aromatic rings are present, then the
rings may be fused together, so that adjacent rings share a common bond.

[0075] The term "isosteric equivalent of a carboxy group" refers to a
group which is physiochemically or topologically similar to carboxylic
acid or carboxylate group. Examples of suitable carboxylic acid or
carboxylate isosteres include, but are not limited to, tetrazole,
tetrazolate, --CONH-tetrazole, oxidiazole, phosphate (--PO3H2),
N-(aryl or heteroaryl)-sulfonamides, acylsulfonamides and sulfonic acid
(--SO3H) (See Patani and LaVoie, 1996 Chem Rev. 96:3147-3176).

[0078] "Heterocyclic" or "heterocyclyl" refers to a non-aromatic ring
having 3 to 8 atoms in the ring and of those atoms 1 to 4 are
heteroatoms, said ring being isolated or fused to a second ring selected
from 3- to 7-membered alicyclic ring containing 0 to 4 heteroatoms,
wherein said heteroatoms are independently selected from O, N and S.
Heterocyclic includes partially and fully saturated heterocyclic groups.
Heterocyclic systems may be attached to another moiety via any number of
carbon atoms or heteroatoms of the radical and may be both saturated and
unsaturated, which includes all forms of carbohydrate moieties.
Non-limiting examples of heterocyclic include pyrrolidinyl, pyrrolinyl,
pyranyl, piperidinyl, piperazinyl, morpholinyl, tetrahydrofuranyl,
tetrahydrothiophenyl, pyrazolinyl, dithiolyl, oxathiolyl, dioxanyl,
dioxinyl, oxazinyl, azepinyl, diazepinyl, thiazepinyl, oxepinyl and
thiapinyl, imidazolinyl, thiomorpholinyl, and the like.

[0101] A particular compound of formula (II) is
3-(4-farnesyloxy-3-methoxyphenyl)propionic acid

##STR00009##

[0102] While some of the compounds of formula (I) or (II) may be obtained
by extraction, others may be obtained by manipulation of isolated
compounds or by synthesis from suitable starting materials.

[0103] For example, compounds of formula (1) or (II) in which Z is oxygen
and R1 is other than farnesyl may be obtained by hydrolysing the
ether farnesyl group and replacing it with another alkyl, alkenyl,
alkynyl or cycloalkyl group.

[0104] The alkenylene chain, Y, may be extended by nucleophilic addition
with a protected carboxy alkyl group to the carboxy group R3
followed by reduction and elimination of the resulting hydroxyl group,
deprotection of the protected carboxyalkyl group may then be performed.

[0105] A person skilled in the art would be able to determine suitable
conditions for obtaining derivatives of isolated compounds, for example,
by reference to texts relating to synthetic methodology, examples of
which are Smith M. B. and March J., March's Advanced Organic Chemistry,
Fifth Edition, John Wiley & Sons Inc., 2001 and Larock R. C.,
Comprehensive Organic Transformations, VCH Publishers Ltd., 1989.
Furthermore, selective manipulations of functional groups may require
protection of other functional groups. Suitable protecting groups to
prevent unwanted side reactions are provided in Green and Wuts,
Protective Groups in Organic Synthesis, John Wiley & Sons Inc., 3rd
Edition, 1999.

[0106] In a further aspect of the invention there is provided a method of
treating or preventing a helminthic infection in a subject comprising
administering an effective amount of an extract from an Acronychia
species.

[0108] In another aspect of the invention there is provided a method of
treating or preventing a cell proliferative disorder comprising
administering to a subject an effective amount of an extract obtained
from an Acronychia species.

[0109] In some embodiments, the cell proliferative disorder is a cancer,
especially where the cancer is selected from leukaemia, melanoma,
prostate cancer, breast cancer, ovarian cancer, basal cell carcinoma,
squamous cell carcinoma, fibrosarcoma, colon cancer, lung cancer, a
neoplasm and other solid tumour cancers. In other embodiments, the cell
proliferative disorder is a non-malignant, for example, benign prostatic
hyperplasia.

[0110] In yet another embodiment of the present invention there is
provided a method of treating or preventing an inflammatory disorder
comprising administering to a subject an effective amount of an extract
from and Acronychia species.

[0111] In some embodiments of this aspect the inflammatory disorder is
general inflammation, rheumatoid arthritis, colitis, bacterial sepsis or
a disorder associated with a malfunctioning immune system, such as an
autoimmune disorder. In some embodiments, the inflammatory disorder is
bacterial sepsis. In some embodiments, the compound of the invention is
capable of immunomodulation, especially immunosuppression. The compounds
of the invention are also useful as immunosuppressive agents in organ
transplantation.

[0112] In another aspect there is provided a method of treating or
preventing a disease or disorder related to oxidative stress comprising
administering to a subject an effective amount of an extract from an
Acronychia species.

[0113] In some embodiments the disease or disorder related to oxidative
stress is a disease or disorder which a subject may benefit from taking
antioxidants. For example antioxidants are beneficial in treating or
preventing cardiovascular disease, such as heart failure, heart attack
and stroke; infectious diseases such as HIV/AIDS, and hepatitis; cancer;
aging diseases such as arthritis, macular degeneration, glaucoma and
cataracts; Lung diseases such as emphysema, chronic obstructive pulmonary
disease (COPD) and adult respiratory distress syndrome; and neurological
or neurodegenerative disorders such as Alzheimer's disease, Parkinson's
disease, multiple sclerosis, Huntington's disease, dementia and cognitive
decline, especially cognitive decline in companion animals such as dogs.

[0114] In some embodiments, the extract from Acronychia species is an
anti-inflammatory antioxidant composition. This is particularly useful
for the treatment of neurological disorders such as Alzheimer's disease.

[0115] In some embodiments the extract may be in the form of a
nutraceutical composition that boosts the subject's antioxidant defences.
The nutraceutical composition may be taken as a supplement while the
subject is suffering from a disease or disorder or may be taken as a
preventative measure to prevent or delay onset of a disease or disorder
related to oxidative stress.

[0116] The extract of Acronychia species may be administered to any
subject in need of treatment for a bacterial infection, a cell
proliferative disorder, an inflammatory disease or a disease or disorder
related to oxidative stress or to a subject who is in need of prevention
of any of these disorders. The term "subject" as used herein includes
humans, primates, livestock animals (eg. sheep, pigs, cattle, horses,
donkeys), laboratory test animals (eg. mice, rabbits, rats, guinea pigs),
companion animals (eg. dogs, cats), birds (eg. chickens, ducks, geese,
parrots, cockatoos, pigeons, finches, raptors, ratites, quail, canaries),
captive wild animals (eg. foxes, kangaroos, deer) and reptiles (eg.
lizards and snakes). In some embodiments, the subject is human, a
companion animal, a livestock animal or a laboratory test animal. In
particular embodiments, the subject is a human, a companion animal or
livestock animal.

[0117] An effective amount of the extract will be dependent on the
extraction procedure and the source of the extract, that is, the
Acronychia species used and whether the extract contains predominantly
one compound or contains many compounds. An "effective amount" means an
amount necessary at least partly to attain the desired response, or to
delay the onset or inhibit progression or halt altogether, the onset or
progression of a particular condition being treated. The amount varies
depending upon the health and physical condition of the individual to be
treated, the taxonomic group of individual to be treated, the degree of
protection desired, the formulation of the composition, the assessment of
the medical situation, and other relevant factors. It is expected that
the amount will fall in a relatively broad range that can be determined
through routine trials. An effective amount in relation to a human
patient, for example, may lie in the range of about 0.1 ng per kg of body
weight to 1 g per kg of body weight per dosage. The dosage is preferably
in the range of 1 μg to 1 g per kg of body weight per dosage, such as
is in the range of 1 mg to 1 g per kg of body weight per dosage. In one
embodiment, the dosage is in the range of 1 mg to 500 mg per kg of body
weight per dosage. In another embodiment, the dosage is in the range of 1
mg to 250 mg per kg of body weight per dosage. In yet another embodiment,
the dosage is in the range of 1 mg to 100 mg per kg of body weight per
dosage, such as up to 50 mg per kg of body weight per dosage. In yet
another embodiment, the dosage is in the range of 1 μg to 1 mg per kg
of body weight per dosage. Dosage regimes may be adjusted to provide the
optimum therapeutic response. For example, several divided doses may be
administered daily, weekly, monthly or other suitable time intervals, or
the dose may be proportionally reduced as indicated by the exigencies of
the situation.

[0118] Reference herein to "treatment" and "prophylaxis" is to be
considered in its broadest context. The term "treatment" does not
necessarily imply that a subject is treated until total recovery.
Similarly, "prophylaxis" does not necessarily mean that the subject will
not eventually contract a disease condition. Accordingly, treatment and
prophylaxis include amelioration of the symptoms of a particular
condition or preventing or otherwise reducing the risk of developing a
particular condition. The term "prophylaxis" may be considered as
reducing the severity or onset of a particular condition. "Treatment" may
also reduce the severity of an existing condition.

[0119] In another embodiment there is provided a use of an extract from an
Acronychia species in the manufacture of a medicament for treating or
preventing a bacterial infection, a helmintic infection, a cell
proliferative disorder, an inflammatory disease or disorder or a disorder
related to oxidative stress.

[0120] In yet another embodiment there is provided a use of a compound of
formula (I):

##STR00010##

[0121] wherein A is an aryl group or a heteroaryl group;

[0122] Z is selected from --O--, --S-- and --NR4--;

[0123] Y is selected from a covalent bond and --(CH2)p--;

[0124] R1 is selected from --C5-C20alkyl,
--C5-C20alkenyl, --C5-C20alkynyl and
--C3-C8cycloalkyl;

[0126] R3 is selected from --CO2H or an isosteric equivalent of
a carboxy group;

[0127] R4 is selected from hydrogen and --C1-C6alkyl;

[0128] and p is an integer from 1 to 10; or a pharmaceutically salt
thereof

[0129] in the manufacture of a medicament for treating or preventing a
bacterial infection.

[0130] In some embodiments the extract may be incorporated into a
nutraceutical composition intended to improve the health and wellbeing of
a subject. In some embodiments the nutraceutical composition is an
antioxidant nutraceutical composition.

[0131] While it is possible that the extract may be used in a method in
neat form, for example, as a dried powder, it is possible that the
extract is formulated in a pharmaceutical or nutraceutical composition
with a pharmaceutically acceptable carrier, diluent and/or excipient.

[0132] Dosage form and rates for pharmaceutical use and compositions are
readily determinable by a person of skill in the art.

[0133] Dosage forms include tablets, dispersions, suspensions, injections,
solutions, syrups, troches, capsules, suppositories, aerosols,
transdermal patches and the like. These dosage forms may also include
injecting or implanting devices designed specifically for, or modified
to, controlled release of the pharmaceutical composition. Controlled
release of the therapeutic agent may be effected by coating the same, for
example, with hydrophobic polymers including acrylic resins, waxes,
higher aliphatic alcohols, polylactic and polyglycolic acids and certain
cellulose derivates such as hydroxypropylmethyl cellulose. In addition,
the controlled release may be affected by using other polymer matrices,
liposomes and/or microspheres.

[0134] Pharmaceutically acceptable carriers for systemic administration
may also be incorporated into the compositions of this invention.

[0135] Suitably, the pharmaceutical composition comprises a
pharmaceutically acceptable excipient. By "pharmaceutically acceptable
excipient" is meant a solid or liquid filler, diluent or encapsulating
substance that may be safely used in systemic administration. Depending
upon the particular route of administration, a variety of carriers, well
known in the art may be used. These carriers or excipients may be
selected from a group including sugars, starches, cellulose and its
derivates, malt, gelatine, talc, calcium sulphate, vegetable oils,
synthetic oils, polyols, alginic acid, phosphate buffered solutions,
emulsifiers, isotonic saline, and pyrogen-free water.

[0136] Any suitable route of administration may be employed for providing
a human or non-human with the pharmaceutical composition of the
invention. For example, oral, rectal, parenteral, sublingual, buccal,
intravenous, intraarticular, intra-muscular, intra-dermal, subcutaneous,
inhalational, intraocular, intraperitoneal, intracerebroventricular,
transdermal and the like may be employed.

[0137] Pharmaceutical compositions of the present invention suitable for
administration may be presented in discrete units such as vials,
capsules, sachets or tablets each containing a predetermined amount of
one or more pharmaceutically active compounds of the invention, as a
powder or granules or as a solution or a suspension in an aqueous liquid,
a non-aqueous liquid, an oil-in-water emulsion or a water-in-oil
emulsion. Such compositions may be prepared by any of the method of
pharmacy but all methods include the step of bringing into association an
extract or one or more pharmaceutically active compounds of the invention
with the carrier which constitutes one or more necessary ingredients. In
general, the compositions are prepared by uniformly and intimately
admixing the agents of the invention with liquid carriers or finely
divided solid carriers or both, and then, if necessary, shaping the
product in to the desired presentation.

[0138] In powders, the carrier is a finely divided solid which is in a
mixture with the finely divided active component.

[0139] In tablets, the active component is mixed with the carrier having
the necessary binding capacity in suitable proportions and compacted in
the shape and size desired.

[0140] The powders and tablets preferably contain from five or ten to
about seventy percent of the extract or active compound. Suitable
carriers are magnesium carbonate, magnesium stearate, talc, sugar,
lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose,
sodium carboxymethylcellulose, a low melting wax, cocoa butter, and the
like. The term "preparation" is intended to include the formulation of
the active compound with encapsulating material as carrier providing a
capsule in which the active component or extract, with or without
carriers, is surrounded by a carrier, which is thus in association with
it. Similarly, cachets and lozenges are included. Tablets, powders,
capsules, pills, cachets, and lozenges can be used as solid forms
suitable for oral administration.

[0141] Liquid form preparations include solutions, suspensions, and
emulsions, for example, water or water-propylene glycol solutions. For
example, parenteral injection liquid preparations can be formulated as
solutions in aqueous polyethylene glycol solution.

[0142] The extract or compounds may thus be formulated for parenteral
administration (e.g. by injection, for example bolus injection or
continuous infusion) and may be presented in unit dose form in ampoules,
pre-filled syringes, small volume infusion or in multi-dose containers
with an added preservative. The compositions may take such forms as
suspensions, solutions, or emulsions in oily or aqueous vehicles, and may
contain formulatory agents such as suspending, stabilising and/or
dispersing agents. Alternatively, the active ingredient may be in powder
form, obtained by aseptic isolation of sterile solid or by lyophilisation
from solution, for constitution with a suitable vehicle, e.g. sterile,
pyrogen-free water, before use.

[0143] Aqueous solutions suitable for oral use can be prepared by
dissolving the active component or extract in water and adding suitable
colorants, flavours, stabilizing and thickening agents, as desired.

[0144] Aqueous suspensions suitable for oral use can be made by dispersing
the finely divided active component or extract in water with viscous
material, such as natural or synthetic gums, resins, methylcellulose,
sodium carboxymethylcellulose, or other well known suspending agents.

[0145] Also included are solid form preparations which are intended to be
converted, shortly before use, to liquid form preparations for oral
administration. Such liquid forms include solutions, suspensions, and
emulsions. These preparations may contain, in addition to the active
component or extract, colorants, flavours, stabilizers, buffers,
artificial and natural sweeteners, dispersants, thickeners, solubilizing
agents, and the like.

[0146] For topical administration to the epidermis the extract or
compounds may be formulated as ointments, creams or lotions, or as a
transdermal patch. Ointments and creams may, for example, be formulated
with an aqueous or oily base with the addition of suitable thickening
and/or gelling agents. Lotions may be formulated with an aqueous or oily
base and will in general also contain one or more emulsifying agents,
stabilizing agents, dispersing agents, suspending agents, thickening
agents, or colouring agents.

[0147] Formulations suitable for topical administration in the mouth
include lozenges comprising active agent or extract in a flavored base,
usually sucrose and acacia or tragacanth; pastilles comprising the active
ingredient in an inert base such as gelatin and glycerin or sucrose and
acacia; and mouthwashes comprising the active ingredient in a suitable
liquid carrier.

[0148] Solutions or suspensions are applied directly to the nasal cavity
by conventional means, for example with a dropper, pipette or spray. The
formulations may be provided in single or multidose form. In the latter
case of a dropper or pipette, this may be achieved by the patient
administering an appropriate, predetermined volume of the solution or
suspension. In the case of a spray, this may be achieved for example by
means of a metering atomising spray pump. To improve nasal delivery and
retention the compounds or extract may be encapsulated with
cyclodextrins, or formulated with their agents expected to enhance
delivery and retention in the nasal mucosa.

[0149] Administration to the respiratory tract may also be achieved by
means of an aerosol formulation in which the active ingredient or extract
is provided in a pressurized pack with a suitable propellant such as a
chlorofluorocarbon (CFC) for example, dichlorodifluoromethane,
trichlorofluoromethane, or dichlorotetrafluoroethane, carbon dioxide, or
other suitable gas. The aerosol may conveniently also contain a
surfactant such as lecithin. The dose of drug may be controlled by
provision of a metered valve.

[0150] Alternatively the active ingredients may be provided in the form of
a dry powder, for example a powder mix of the compound or extract in a
suitable powder base such as lactose, starch, starch derivatives such as
hydroxypropylmethyl cellulose and polyvinylpyrrolidone (PVP).

[0151] Conveniently the powder carrier will form a gel in the nasal
cavity. The powder composition may be presented in unit dose form for
example in capsules or cartridges of, e.g., gelatin, or blister packs
from which the powder may be administered by means of an inhaler.

[0152] In formulations intended for administration to the respiratory
tract, including intranasal formulations, the compound or extract will
generally have a small particle size for example of the order of 1 to 10
microns or less. Such a particle size may be obtained by means known in
the art, for example by micronization.

[0153] In yet another aspect of the invention, there is provided the use
of an extract from an Acronychia species in the manufacture of a
medicament for treating or preventing a bacterial infection, a cell
proliferative disorder, an inflammatory disease or disorder or a disease
or disorder related to oxidative stress.

[0154] In another aspect of the invention the extract from Acronychia
species may be incorporated into a cosmetic composition for its
antibacterial, anti-inflammatory and/or antioxidant properties.

[0156] Formulation of cosmetic compositions including antioxidant and/or
antibacterial components is known in the art.

[0157] In a further aspect of the invention there is provided a cosmetic
composition comprising an extract from an Acronychia species, wherein the
extract is obtained by a method comprising initial water or alcohol
extraction and a subsequent ethyl acetate extraction.

[0158] In another aspect of the invention, the extract of an Acronychia
species is used as a food additive, a fragrance component or an
antioxidant or antibacterial component of a cosmetic composition.

[0159] The extracts from Acronychia species have volatile components which
provide flavour and fragrances. These volatile components may be used as
a food additive in foods such as jams, ice cream, confectionary and
sauces. Alternatively, the extract containing volatile components may be
used as a fragrance in perfumes, cosmetics, household products such as
cleaners, deoderisers or fabric softeners or in personal care products
such as talcum powder, deodorants, soaps, shampoos, conditioners and the
like.

[0160] The formulation of such household products or personal care
products containing fragrances is well known in the art. For example, the
extract may be included in the household product or personal care product
in an amount in the range of 2-5%.

[0161] A number of volatile compounds were isolated from the extract of A.
acidula as shown in FIG. 1. Compounds identified include
1-ethenyl-1-methyl-2,4-bis(1-methylethenyl)cyclohexane,
2,6-di-tert-butylbenzoquinone, 2,5-di-tert-butyl-1,4-benzoquinone,
Tetracontane-1,40-diol, and
2,2,5,5-tetramethyl-bicyclo[6.3.0]undec-1(8)-enone. Smaller amounts of
unidentified volatile components were also found by mass spectrometry.

[0163] In another aspect. of the present invention there is provided a
flavour or fragrance composition comprising one or more of the following
compounds: [0164]
1-ethenyl-1-methyl-2,4-bis(1-methylethenyl)cyclohexane, [0165]
2,6-di-tert-butylbenzoquinone, [0166] 2,5-di-tert-butyl-1,4-benzoquinone,
[0167] Tetracontane-1,40-diol, and [0168]
2,2,5,5-tetramethyl-bicyclo[6.3.0]undec-1(8)-enone.

[0169] In some embodiments, the flavour or fragrance composition contains
at least two of the components listed above. In some embodiments the
flavour or fragrance composition contains all of the components listed
above.

[0171] In order that the invention be readily understood and put into
practical effect, particular embodiments will now be described by way of
the following non-limiting examples.

EXAMPLES

[0172] Screening Procedures

[0173] Antioxidant Activity

[0174] DPPH Assay for radical scavenging

[0175] Background

[0176] This assay is based upon the use of the stable free radical DPPH,
which is a deep violet colour. When a solution of DPPH is mixed with that
of a substance that can donate a hydrogen atom (e.g. an antioxidant), it
gives rise to the reduced form of DPPH, with the loss of this violet
colour.

[0177] Materials

[0178] 2,2-Diphenyl-1-picryl-hydrazyl (DPPH); MW:394

[0179] Ethanol

[0180] Sample to be assayed at dilutions required

[0181] VERSAmax tunable microplate reader--Molecular Devices

[0182] Method [0183] 1. A 0.1 mM solution of DPPH in ethanol (3.94 mg of
DPPH in 100 mL of Ethanol) was prepared. Alternatively (If serial
dilutions are to be done in the plate) a 10× solution of DPPH (1
mM, 3.94 mg in 10 mL ethanol) was prepared. Solutions were stored at
4° C. for a few weeks but lose colour over time. [0184] 2. 90
μL of 1× DPPH solution was added to each well used in the
96-well plate. [0185] 3. 10 μL of sample to be assayed was added to
the wells containing DPPH (for a final dilution of 1/10) or 10 μL of a
dilution of sample. Each sample was assayed in triplicate. A negative
control of 10 μL ethanol was used replacing the sample. A positive
control of a known concentration of catechin was used if required. If
samples were coloured, control wells of ethanol and 10 μL of sample
were used as colour blanks. [0186] 4. If serial dilutions of sample are
performed in the plate, 90 μL of ethanol was added to the wells. 10
μL of sample was added to the top well and serially diluted 10 μL
down the plate, removing 10 μL from the last well diluted to ensure an
equal amount in each well. 10 μL of 10× DPPH, was added to each
well. [0187] 5. After combining sample and DPPH, plates were left at room
temp for 20 minutes before reading on the plate reader at a wavelength of
517 nm.

[0188] Lipid Peroxidation in Human Plasma

[0189] Background

[0190] Inhibition of lipid peroxidation is a major target for
antioxidants. It has been shown by Itoh et al. 2004, Biochemical
Pharmacology, 68, 813-818 (and references cited therein) that the free
radical-mediated oxidation of human plasma in vitro generates cholesteryl
ester hydroperoxide (CEOOH) as a major product, with smaller amounts of
cholesteryl ester hydroxide (CEOH) and phospholipid hydro(per)oxide.
Consequently, the extent of lipid peroxidation in plasma can be estimated
from the formation of cholesteryl ester hydroperoxide (CEOOH).

[0191] Method

[0192] The effect of samples in reducing lipid peroxidation in dialysed
human plasma was performed following the method of Itoh et al. 2004.
Briefly this involved: [0193] 1. Blood was collected in
ethylenediaminetetraacetic acid (EDTA) containing tubes from healthy
volunteer after overnight fasting. Plasma was obtained by centrifugation
at 1580×g for 10 min at 4° C. The plasma was dialyzed using
a dialysis membrane for 18 h at 4° C. in PBS to eliminate ascorbic
acid and other water-soluble small molecular weight antioxidants that
were present. [0194] 2. Lipophilic
2,2'-azobis-(4-methoxy-2,4-dimethylvaleronitrile) (MeO-AMVN) (final
concentration 0.2 mM) was added to PBS solution containing 10 volume % of
dialyzed human plasma with and without 6.66 volume % of sample solution
and the mixture was incubated at 37° C. for 1 hr. [0195] 3. Lipid
peroxidation products were extracted with chloroform/methanol (2/1 by
volume) and the chloroform layer analyzed. Cholesteryl ester
hydroperoxide (CEOOH) and cholesteryl ester hydroxide (CEOH) were
measured at 234 nm by HPLC with a spectrophotometric detector using an
ODS column (Wakosil-II 5C18RS, 5 um, 250 mm×4.6 mm) with
acetonitrile/isopropyl alcohol/water (44/54/2 by volume) eluted at 1
mL/min.

[0201] All antibacterial assays were carried out in a laminar flow cabinet
using the protocol described below. Growth or no-growth was assessed
visually and by ELISA reader. The endpoint is reported as the minimum
dilution (calculated also in μg/mL for the more potent fractions)
required to totally inhibit growth over the 24 hr treatment period.

[0202] Method [0203] 1) A starter culture was set up by scraping a tip
through a frozen aliquot of bacterial culture and placing in 10 mL of
Columbia broth within a falcon tube. This was then placed in the
bacterial shaker at 37° C. at 210 rpm overnight. [0204] 2) A blank
reading was made on the spectrophotometer at 600 nm using 210 of Columbia
broth. A reading of 200 μL of the starter culture was then taken.
[0205] 3) In calculations, a final concentration of 0.005 was desired.
The formula used for dilution of x mL starter was: x*A600=0.005*desired
volume [0206] 4) 90 uL of MeOH was added to each well of a 96 well flat
bottomed plate. 10 μL of sample was added to the top well and diluted
1:10 down the plate. Alternatively the top rows were left empty of MeOH
and 100 μL of sample was added and then diluted at 1:10 down the
plate. [0207] 5) The plates were then left to dry in a biological safety
cabinet for 2-3 hours or until all solvent had evaporated off the plate.
[0208] 6) 100 μL of diluted bacterial culture was then added to each
well of the plate. The plate was then placed in the bacterial incubator
at 37° C. overnight. The following day the plate was scored under
the microscope for inhibition (clear solution indicating 100% inhibition
compared with turbid solution for controls) and aggregation. For
quantitation, plates were read at 405 nm (ELISA).

[0209] Nematicidal Screening

[0210] An anthelmintic bioassay, applicable to all parasitic nematodes
with free-living life cycle stages, was used as a screen to detect
activity and potency of crude extracts from Acronychia species against
parasitic nematodes. The assay determines the effects of test extracts on
larval development and follows the method described by Gill et al. (1995)
Int. J. Parasitol. 25: 463-470.

[0211] Briefly, in this assay eggs of the nematode Haemonchus contortus
(McMaster strain) were applied to the surface of an agar matrix
containing the test sample and allowed to develop through to the L3,
infective stage (6 days). At this time the stage of larval development
reached and any unusual features (deformity, paralysis, toxicity) were
noted by microscopic examination. To determine potency of the extracts, a
series of two-fold dilutions of the parent extract are used and the,
readout from this assay is a dilution titre.

[0212] Anti-Inflammatory and Neuroprotectant Assay

[0213] Background

[0214] Production of nitric oxide and the pro-inflammatory cytokine TNF
are associated with oxidative stress and inflammatory processes in
general, and are increasingly implicated in the development of
age-related neurological conditions, including Alzheimer's disease. An
assay system to measure LPS or interferon-γ induced production of
nitric oxide in N-11 murine microglial cells can be used to identify
fractions and compounds with anti-inflammatory, neuroprotective and
antioxidant activity. The "gold standards" for comparison in this assay
are the bioflavonoids, apigenin and diosmetin.

[0219] Once the cells had grown to confluence within the culture flask,
they were removed using a rubber policeman, as opposed to trypsin
treatment which is known to be responsible for removing membrane-bound
receptors such as RAGE. The cell suspension was then concentrated by
centrifugation for 3 minutes at 900×g and resuspended in a small
volume of DMEM containing 0.1% FBS.

[0220] Cell Count

[0221] Equal volumes of resuspended cell solution and Trypan blue (0.1%)
were mixed to a total volume of 20 μL. Half of the mix was placed onto
a Neubauer counting slide. Sixteen squares of equal size were viewed and
counted under a microscope. The number of cells per microliter of cell
solution was calculated, by using the following equation: cell
count×2(dilution factor for Trypan blue)×10(1/100 of total
volume counted)=number of cells per microliter.

[0222] Dispensing of Cells into Plates

[0223] Once the concentration of cell suspension was determined, the
volume of media was adjusted to the required concentration. This was
achieved by dividing total number of cells required by the number of
cells per microliter. 100 μL of cell suspension was administered to
each well of a 96-well plate to give a desired cell total of
5×104 cells per well. For experiments using 6-well plates,
1.2×106 cells per well were distributed into each well with a
final total volume of 2 mL per well. For experiments using 24-well
plates, 3×105 cells per well were distributed into each well
with a final total volume of 1 mL per well. Plates were centrifuged at
500×g for 5 minutes to allow even distribution.

[0224] Activation of Cells

[0225] Following the dispensing of cells into plates, plates were
incubated overnight at 37° C. to allow for settlement and
attachment to the bottom of the wells. Before activation, conditioned
media was replaced with fresh DMEM containing 0.1% FBS to minimize the
effect, of growth during experimentation.

[0226] Lipopolysaccharide

[0227] A stock solution of 1000 μg/mL of E. coli LPS (serotype 0127:B8)
in sterile PBS was used for cell dilutions. Concentrations of LPS ranging
from 0.1 μg/mL to 100 μg/mL were used for the construction of a
dose-dependent activation curve. A concentration of 10 μg/mL was used
to activate cells in assays with extract's.

[0228] Interferon-γ

[0229] A stock solution of 1000 U/mL of murine IFN-γ (Lot #10098)
was used in conjunction with DMEM with 0.1% FBS. A dose-dependent
activation curve was constructed using concentrations of IFN-γ
ranging from 0.1 U/mL to 500 U/mL. A concentration of 10 U/mL was used to
activate cells in assays with extracts.

[0230] Activation of Cells for Assays with Extracts

[0231] Due to the inconsistent nature of LPS in activating cells, a
combination of 10 μg/mL of LPS and 10 U/mL of IFN-γ was used to
activate cells for assays with extracts.

[0232] Nitric Oxide Determination

[0233] Nitric oxide was determined by the Griess reagent quantification of
nitrite, one of its stable reaction products. The Griess reagent was made
up of equal volumes of 1% sulfanilamide and 0.1% napthyethylene-diamine
in 5% HCl, and in the presence of nitrite forms a violet colour. A
standard curve using known concentrations of sodium nitrite was
constructed, and the absorbance of each sample was measured at 545 nm
using a Wallac VICTOR3 V Multilabel Counter (Perkin Elmer). 75 μL
of supernatant from each activated well was transferred to a fresh
96-well plate and mixed with equal volumes of Griess reagent. Absorbance
was measured, enabling the calculation of nitrite concentration from the
sodium nitrite standard curve.

[0234] Alamar Blue Cell Viability Assay

[0235] Alamar blue is a fluorescent redox indicator used to measure cell
viabilities. It involves the cellular reduction of resazurin by cellular
mitochondrial enzymes, yielding a soluble product (resorufin), which is
directly proportional to cell number. Cells were incubated with 100 μL
per well of a 1% resazurin and DMEM (0.1% FBS) solution for 2 hours at
37° C. in 96-well plates. The total volume was adjusted for
24-well plates so that each well was incubated with 500 μL.
Fluorescence was examined at 545/595 nm and expressed as a percentage of
the control cells, following the subtraction of background readings.

[0241] The normal cells are primary human fibroblasts (NFF). The tumour
cell lines used are MM418c5 melanoma (a pigmented cell line, to detect
depigmenting agents), MCF-7 breast cancer, CI80-13S ovarian cancer
(cisplatin-resistant; highly sensitive to mitochondrial drugs) and LnCAP
prostate cancer line. [0242] 1. The cells are sparsely seeded and
allowed to grow in tissue culture medium (RPMI1640+10% FCS+pen/strep) for
5-7 days to provide clonogenic type cell survival data. [0243] 2. The
amount of cell suspension required to give 4000 cells per volume of 100
μL was calculated. This required cell suspension was mixed with tissue
culture medium (with tyrosine added) to make up to the required volume.
[0244] 3. The cell suspension was plated out at 100 μL per well into
flat-bottomed 96-well tissue culture plates, before being placed into a
37° C., 5% CO2, humidified incubator overnight. [0245] 4. 2
μL of the required fraction was then placed into the top well (row A)
of a plate and mixed using a pipette before being serially diluted 1:10
down the plate to row G. [0246] 5. The plates were incubated again at
37° C. for 3-4 days before being assessed under a light microscope
for cellular viability and morphological changes, particularly
depigmentation and hyper-pigmentation of cells.

[0247] Preliminary Cell Screening of S1/S2 Fractions

[0248] Method for Mixed Cell Plates [0249] 1. Four different cell lines
[NFF, MCF7, MM96L (melanoma) and K562 (leukemia)] suspended in tissue
culture medium (RPMI1640+10% ECS+pen/strep) were counted. [0250] 2. The
amount of cell suspension required to give 1000 MM96L, 2000 K562, 3000
MCF7, and 5000 NFF per volume of 100 μL was calculated. This required
cell suspension from each cell line was mixed together and tissue culture
medium added to make up to the required volume. [0251] 3. The mixed cell
suspension was plated out at 100 μL per well into flat-bottomed
96-well tissue culture plates, before being placed into a 37° C.,
5% CO2, humidified incubator overnight. [0252] 4. 2 μL of the
required sample was then placed into the top well (row A) of a plate and
mixed using a pipette before being serially diluted 1:10 down the plate
to row G. [0253] 5. The plates were incubated again at 37° C. for
3-4 days before being assessed under a light microscope for cellular
viability and morphological changes.

[0254] Method for MM418c5 Cell Plates [0255] 6. The MM418c5 melanoma
cell line suspended in tissue culture medium (RPMI1640+10%
FCS+pen/step+250 μg/mL Tyrosine) was counted. [0256] 7. The amount of
cell suspension required to give 4000 cells per volume of 100 μL was
calculated. This required cell suspension was mixed with tissue culture
medium (with tyrosine added) to make up to the required volume. [0257] 8.
The cell suspension was plated out at 100 μL per well into
flat-bottomed 96-well tissue culture plates, before being placed into a
37° C., 5% CO2, humidified incubator overnight. [0258] 9. 2
μL of the required sample was then placed into the top well (row A) of
a plate and mixed using a pipette before being serially diluted 1:10 down
the plate to row G. [0259] 10. The plates were incubated again at
37° C. for 3-4 days before being assessed under a light microscope
for cellular viability and morphological changes, particularly
depigmentation and hyper-pigmentation of cells.

[0260] Definition of Flavour and Fragrance Components

[0261] Background

[0262] Solid-phase microextraction (SPME) is a sample preparation and
sample introduction method in which analytes partition from a sample into
a polymer, coated on a fused silica rod of typically 1 cm length by 100
μm diameter. The fibre is fastened into the end of a fine stainless
steel tube contained in a syringe-like device, and protected by an outer
stainless steel needle. The device's plunger is depressed to expose the
fibre to the sample matrix, retracted at the end of the sampling time,
and then depressed again to expose the fibre to a desorption interface
for analysis by GC-MS. SPME provides an alternative to headspace GC for
analysis of volatiles in a wide range of situations (Pawlisyzn J 1999:
Applications of Solid Phase Microextraction, Royal Society of Chemistry).

[0263] Method

[0264] Frozen fruit of Acronychia acidula was chopped into approximately 5
mm cubes and gently bruised in a mortar and pestle to disrupt the outer
cells and to release volatile components. The chopped fruit was then
transferred immediately to a glass container fitted with a septum. A
commercial SPME fibre holder (Supelco Analytical, USA) and fibre
(polydimethylsiloxane, 100 μm film 57310-U) was inserted through the
septum and the plunger used to lower and expose the fibre to the volatile
mixture in the glass container for 15 minutes. The fibre was then
retracted and later desorbed and analysed by GC-MS.

Example 1

Crude Extract Preparation

[0265] Frozen fruit of Acronychia acidula was sliced into approximately 5
mm cubes, generously covered with ethanol (˜2 L) and shaken
overnight. This extract was then filtered and most of the solvent removed
by rotary evaporation to afford an aqueous concentrate (the crude
extract).

Example 2

First Fractionation Using a Silica Column (S1)

[0266] Preparation of Extract for S1 Column [0267] 1. 200 mL to 1 L of
milli-Q water was added to dried or concentrated crude extract (extract
should have all solvents apart from water dried off, the volume of water
used was dependant on amount of extract) [0268] 2. The same volume of
ethyl acetate was added to the sample and mixed before transferring to a
separating funnel. This is shaken for ˜30 seconds, with care taken
to release the pressure in the funnel frequently. [0269] 3. The two
layers were allowed to separate then collected separately. [0270] 4. The
water layer was placed back into the funnel then steps 2-3 repeated.
[0271] 5. A 1 mL sample from each layer was collected and stored, then
the individual layers dried on the rotary evaporator and weighed. [0272]
6. The ethyl acetate layers were combined and dissolved in a small amount
of ethyl acetate solvent before being stored at 4° C. or
-20° C. until required for silica column chromatography.

[0273] Method for Pilot Silica Column (S1)

[0274] Column Preparation: [0275] 1. 1 g of Merck Silica Gel 60
(0.063-0.2 mm) was weighed into a 100 mL beaker. [0276] 2. Silica was
covered with ˜10 mL of petroleum spirit and mixed thoroughly with a
spatula. [0277] 3. The silica was poured directly after stirring into a
glass column of 1 cm internal diameter, the tap of the column was opened
and the silica allowed to run through for a couple of minutes. [0278] 4.
The tap was turned off and the silica allowed to settle for at least one
hour (preferably overnight). The top of the column was covered with an
air-tight seal to prevent solvent from evaporating. [0279] 5. After
settling, any bubbles in the column were gently tapped out. If any large
air-bubbles were present, the entire bed was stirred up and allowed to
re-settle. [0280] 6. The height of the column was recorded (approx. 3
cm).

[0281] Sample Preparation: [0282] 7. Approximately 150 mg of the
appropriate concentrated crude extract was measured into a small round
bottom flask. [0283] 8. The sample was dried by rotary evaporation and
the weight recorded. [0284] 9. The dried sample was re-dissolved in ethyl
acetate in a volume equal to 2 times the mass of the sample. [0285] 10.
Petroleum spirit was then added to the sample in small volumes (20-50
μL) until the sample started to precipitate out of solution. The
precipitate was re-dissolved by adding a small amount of ethyl acetate.
The volume of pet spirit added was recorded. The load volume should
always be under 2 mL. [0286] 11. A 200 μL pipette was used to
accurately measure out 1/6th of the of the load (˜20 mg). This
was placed in the last well in a set of 24 wells (two rows) of a 2 mL 96
well plate (polypropylene, chemically resistant).

[0287] Sample Fractionation: [0288] 12. 4 mL of each of the following
solvent ratios was prepared as shown in Table 1:

[0289] 13. The solvent in the column was allowed to elute until the
level of the solvent was just above the silica. [0290] 14. The remaining
load was gently run down the sides of the column using a 1 mL Pasteur
pipette, taking care to ensure the load was even. The sides of the column
were then rinsed with a small amount of solvent to remove any load.
[0291] 15. The tap was opened and the load allowed to move onto the
column. [0292] 16. Using a Pasteur pipette as before, the first solvent
mix was added to the column with care taken not to disturb the silica.
[0293] 17. The solvent was allowed to move through the column, and for
each solvent two 2 mL fractions were collected sequentially into the 2 mL
96-well plate. [0294] 18. The above was repeated for each solvent
ensuring that the previous solvent had eluted to the top of the column
before addition of new solvent. [0295] 19. The fractions in the 96-well
plate were dried by blowing nitrogen over them. [0296] 20. 400 μL of
ethanol was added to each dried fraction and mixed using a pipette to
dissolve the samples. 100 μL of each sample was transferred to each of
3 round-bottom 96-well plates.

[0304] The final extract (53 g) was dissolved in EtOAc (150 mL) and silica
(about 106 g) was added in order to prepare a homogeneous suspension.
Hexane (100 mL) was added and the solvents were removed. Residual mixture
was dried at 50° C./20 Torr for 1 h. The column was packed with
silica and the obtained dry load was added to the top of the column (424
g of silica, column diameter 10 cm×13.0 cm and dry load on silica
3.0 cm, total ˜1200 mL), which was eluted, using vacuum suction,
with the solvents indicated in Table 2 and collected fractions as shown
in the Table 3.

[0305] Collected fractions were dried and transferred into 4.5 mL vials as
solutions in a suitable solvent (see Table 3). The analytical samples of
fractions were prepared by dilution of 45 μL amount of fraction
solutions to 1 mL of MeOH. Large fraction (EB1328-S2-4) was left for
fractional crystallization to provide three samples (see Table 3).

[0306] The fractions EB1328-S2-4 (12.42 g), EB1328-S2-13 and EB1328-S2-14
solidified on evaporation and EB1328-S2-4 was re-crystallized from
hexane/acetone to provide three fractions (solid--EB1328-S2-4solid,
impure solid--EB1328-S2-4solid impure, and oil--EB1328-S2-4mL), which
were analysed by GCMS. Fraction EB1328-S2-solid according to GCMS is a
single compound with retention time 28.20 min. Fractions EB1328-S2-4solid
impure and EB1328-S2-4 mL, excluding volatile compounds, consist of four
main components with retention times (Rf) 7.06, 27.96, 28.20, 33.71
min. The major component for both EB1328-S2-4solid-impure and EB1328-S2-4
mL fractions has Rt 27.96 min with concentration about 77% and 63%
respectively.

[0307] The fractions EB1328-S2-13 and EB1328-S2-14 were filtered for
collection and the formed precipitates were washed with acetone.
According to JH and 13C NMR, the solid collected from both
fractions was citric acid [Olennikov et al. Chem. Nat. Compounds, 2005,
41(4); p 467-468].

[0310] Crude ethanolic extracts of fruit of Acronychia acidula and three
other Acronychia species (A. aberrans, A. acronychioides and A.
crassipetala) were screened in a number of assays to characterize their
antioxidant, antibacterial, anti-inflammatory and anticancer activities
(Table 4). The crude extracts were assayed using DPPH method as an assay
for radical scavenging, Lipid peroxidation assay, Antibacterial growth
assay, and preliminary anticancer screening assays as described above.

[0311] Further studies then focussed on A. acidula as the species for
activity-guided isolation to identify specific `active` fractions in the
fruit because of its availability from small-scale commercial
plantations.

Example 5

Identification of Bioactive Fractions

[0312] Flash chromatography on silica of an ethyl acetate extract of
Acronychia acidula (see S1 method in protocols above) was used as the
first stage in deconvoluting the complex mixture in the crude extract to
identify biologically active fractions. Specific fractions with
moderately potent antibacterial and anticancer activity were identified
(Table 5), while strong anti-inflammatory/antioxidant activity (<0.05
mg mL-1) was present in many of the fractions.

[0314] To more specifically identify fractions and compounds with the
antibacterial activity, 10 g of freeze-dried powder of A. acidula
(EB1328) was extracted three times with petroleum spirit (3×50 mL).
Combined extracts were concentrated to dryness in a rotary evaporator and
resulting residue (0.248 g) was dissolved in methanol (HPLC grade) at 10
mg/mL and separated by HPLC. HPLC purification was carried out using
Gracevydac C18 reversed phase column (100×4.60 mm, 120 A) at a flow
rate of 0.5 mL/min of methanol/water (80/20 v/v and then gradient to 100%
of methanol). HPLC fractions (0.5 mL) were collected using Gilson
fraction collector FC204 and tested for antibacterial activity against
Streptococcus salivarius. Results showing fractions active in the
antibacterial test are presented in Table 7.

[0315] To further identify active antibacterial components in A. acidula,
fractions correlating to the three dominant peaks (15.0-18.0, 18.1-20.0
and 20.1-22.8 retention times) were pooled separately. The purity of the
collected HPLC fractions was confirmed by running samples with an
alternate HPLC solvent system (0.5 mL/min of methanol/water 70/30 mL/min)
and by GC-MS analysis. On the basis of HPLC analysis, peak purity was
85-95%.

[0316] Active fraction 22 (coded as EB1328-QL02) was determined by GC-MS
to be pure material with M.sup.+ 370.5. 1H and 13C NMR spectra were
recorded and are shown in Table 8. 1H and 13C NMR proved this
structure to be known compound 3-(4-farnesyloxyphenyl)propionic acid (CAS
No. 126269-87-2); this structure was designated with identifier EBC111.